A novel T cell receptor, TCR gamma delta, is found on a small subset of human T lymphocytes. This receptor is a CD3-associated heterodimer composed of the product of the TCR gamma gene and the product of a newly identified gene, TCR delta. The nature of the antigens and/or restricting elements recognized by TCR gamma delta is unknown. The goal of this proposal is an understanding of the TCR gamma delta repertoire and the forces that mold it, with an emphasis on the TCR delta gene. Initial studies indicated a limited TCR delta germline reperoire, with high junctional diversity. Because the C delta locus is nested within the TCR alpha locus, between V alpha and J alpha gene segments, it becomes imperative to examine more completely the extent of the V delta repertoire, and its relationship to the V alpha repertoire. This will be accomplished through the analysis of TCR delta cDNA clones obtained from clonal and polyclonal TCR gamma delta cell sources. A number of approaches will be used to determine how distinction between the two repertoires is maintained. Genomic cloning, deletion mapping and field inversion gel electrophoresis will be use to map the locations of TCR delta gene segments relative to TCR alpha gene segments, to determine whether gross chromosomal organization is important . Transfection experiments will be used to assess constraints in chain pairing, receptor assembly and function as an explanation for biases in the usage of specific TCR gamma and TCR delta gene segments. A role for cis-acting regulatory sequences in imparting specificity to the process of rearrangement will also be sought. Structural and functional analysis of promoter and enhancer elements will be used to identify candidates for such sequences. In addition, rearrangement specificity will be assessed in a transgenic mouse model system, to allow direct analysis of flanking sequences controlling this process. An understanding of the distinctions between the TCR gamma delta and TCR alpha beta repertoires and how they are maintained will be an important part of the overall approach to reveal the function of this novel receptor system.